[Histonet] Re: DIF tissue in GLUT

Johnson, Teri TJJ <@t> stowers.org
Thu Aug 27 14:02:09 CDT 2009

William, thanks for the reminder, I had totally forgotten about using a counterstain to counteract AF in aldehyde fixed samples. We have some glutaraldehyde fixed cryosections of kidney I think I'll try this with. I can assure you, the autofluorescence in these samples is magnificent! I worry about using sodium borohydride because they are cryosections and I've heard the treatment can be a bit harsh on tissue.

Here's some additional information since we're on the subject: http://www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.pdf

Kind regards,
-----Original Message-----
From: WILLIAM DESALVO [mailto:wdesalvo.cac <@t> hotmail.com]
Sent: Thursday, August 27, 2009 12:51 PM
To: Johnson, Teri; histonet
Subject: RE: [Histonet] Re: DIF tissue in GLUT

I suggest you use a counterstain for your IF to reduce the autofluoresence. Evans Blue - the product can be used as a counterstain in immunohistochemistry when using FITC. After staining for immunofluorescence, dip sections in a 0.1% (w/v) in water solution of Evans Blue for 5-10 minutes. Rinse well in fresh PBS or water before coverslipping. Reference: Immunocytochemistry, Theory and Practice, p. 82 (1988). Purchase from Sigma-Aldrich.

William DeSalvo, B.S., HTL(ASCP)

> From: TJJ <@t> stowers.org
> To: histonet <@t> lists.utsouthwestern.edu
> Date: Thu, 27 Aug 2009 12:18:47 -0500
> Subject: [Histonet] Re: DIF tissue in GLUT
> Anne,
> Tissue that has been fixed in glutaraldehyde has very, VERY bright autofluorescence. Unless there is some way to minimize this (none that I'm aware of), your immunofluorescence will be impossible to read over the background signal.
> Teri Johnson, HT(ASCP)QIHC
> Managing Director Histology Facility
> Stowers Institute for Medical Research
> 1000 E. 50th St.
> Kansas City, MO 64110
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